Abstract
The rates of colorectal cancer (CRC) in Aotearoa, New Zealand, are among the highest internationally, with more than 3,000 individuals diagnosed in New Zealand every year. As New Zealand’s population ages, CRC will likely pose a significant burden on our communities and healthcare system. Therefore, understanding the aetiology of CRC may inform advancements in preventative measures and treatments.
The tumour microenvironment (TME) plays an important role in the initiation and progression of cancers. Tumour cells secrete high numbers of extracellular vesicles (EVs) into the surrounding microenvironment, which can then enter the circulatory system. EVs are essential mediators of local and distant intercellular signalling as they can be internalised by other cells and modulate the function of the recipient cell. Circulating monocytes can encounter tumour derived EVs in the circulatory system or when they extravasate into the TME. In CRC, monocytes and their derivatives, macrophages, can exhibit both tumour-suppressive and tumour-promoting properties. One way in which their fate may be determined is through interactions with CRC-EVs, which may predispose circulating monocytes towards phenotypes that promote a favourable microenvironment for tumour survival. Therefore, this study aimed to investigate the impact of CRC-EVs on gene expression in healthy monocytes.
EVs were isolated from the cell culture media of two CRC cell lines, DLD1 (colon adenocarcinoma) and SW837 (rectal adenocarcinoma), using size exclusion chromatography (SEC). CRC-EVs were characterised using a BCA assay and Western Blotting. Protein concentration was found to be similar in EVs secreted by DLD1 cells (45.97μg/mL ± 3.51μg/mL) and SW837 cells (44.62μg/mL ± 2.03μg/mL). Western blots (CD63 and Calnexin) confirmed the presence and purity of the isolated EVs.
Monocytes were isolated from the whole blood of one healthy volunteer and were co-cultured with CRC-EVs for 24 hours. The effect of CRC-EVs on monocyte metabolic activity was 2 assessed using a CCK-8 assay. Results showed no significant differences in metabolic activity between monocytes treated with 0.25μL to 20μL of DLD1-EVs and SW837-EVs, relative to monocytes treated with “Dummy-EVs”.
To analyse the impact of CRC-EVs on monocyte gene expression, RT-qPCR was conducted to assess the mRNA expression of anti- (IL10, TGFB) and pro-inflammatory (TNF, IL1B, IL6, CXCL10) cytokines, inflammasome proteins and enzymes (ASC, CASP1, CASP5) and pattern recognition receptors (TLR2, TLR4, NOD1, NOD2). The addition of CRC-EVs to healthy monocytes induced changes in gene expression, which were distinct between DLD1-EVs and SW837-EVs, indicating that the immunomodulatory role of CRC-EVs may depend on the cell from which the EVs originate.
The preliminary data presented here provides the basis for future investigations into the immunomodulatory role of patient-derived EVs.